In recent years, peptides from partial enzymatic hydrolysates of food proteins produced in vivo or in vitro have received greater attention from food scientists than ever before. Many biological peptides with health benefits such as opioid activity, antihypertensive activity, antibacterial activity, mineral-binding activity, anti-thrombotic activity, anti-gastric activity, enhancement of intestinal activity, etc have been identified from food protein hydrolysates. These peptides are hidden in a latent state within the sequence of the parent protein and may be released by proteolytic processes during in vivo/in vitro digestion or during food processing. Biologically active peptides derived from daily food proteins would be useful in the development of novel functional food additives and for therapeutic uses.
Hypertension is one of the major independent risk factors for arteriosclerosis, stroke, myocardial infarction and end-stage renal disease. Angiotensin 1-converting enzyme (EC 3.4.15.1, referred to herein as ‘ACE’), which is a dipeptidyl-carboxypeptidase, plays an important physiological role in regulating blood pressure. ACE raises blood pressure by converting an active form of the decapeptide, angiotensin I, to a potent vasoconstrictor octapeptide angiotensin II, the most potent naturally occurring pressor substance known. ACE also degrades vasodilative bradykinin, which has a depressor action. This enzyme also plays a physiological role in the regulation of local levels of other endogenous peptides. For these reasons, specific inhibitors of ACE are useful for regulating blood pressure in the human body. Because ACE activity is closely associated with the development of hypertension and arteriosclerosis, in vitro inhibition of ACE has been used for screening therapeutic agents. Therapeutic vasodepressors such as Captopril and D-2 methyl-3-mercaptopropanoyl-L-proline have been synthesized as ACE inhibitors. Synthetic ACE inhibitors are very potent and have adverse effects that are generally not considered safe.
Interest has recently been focused on the isolation and identification of purified ACE inhibitory peptides from various food sources or food protein hydrolysates after enzymatic digestion. An ACE inhibitor derived from food protein was first reported by Oshima et al., 1979 (Oshima, G., Shimabukuro, H and Nagasawa, K. (1979) “Peptide inhibitors of Angiotensin converting enzyme digests of gelatin by bacterial collagenase”. Biochim. Biophys. Acta, 556, 128). Since then protein digests containing ACE inhibitors have been produced from a variety of edible sources including milk, eggs, chicken, pork beef, fish, and soybean, maize and micro algae (Ariyoshi, Y., (1993), “Angiotensin converting enzyme inhibitors derived from food proteins” Trends Food Sci. Technol., 4, 139–144). Peptides derived from casein and soybean protein have been developed based on the expectation of low toxicity and high safety. Peptides exhibiting ACE-inhibiting activities have been separated from enzymatic hydrolysates of casein (Japanese Laid-Open Patent publication Nos 62-270533, 64-5497, 64-83096) and soybean protein (Japanese Laid-Open Patent publication No. 3-1671981.
Soybean is a source of high quality proteins all over the world. Defatted soy meal contains nearly 50–55% protein. The major proteins of soybeans are the storage globulin classified as 2S, 7S, 11S and 15S protein based on sedimentation coefficients. The 11S fraction also known as glycinin constitutes 25–35% of the total proteins (Liu 1997, In: Soybeans: Chemistry, Technology and Utilization, pp 25–113, Chapman & Hall, New York).
Reference may be made, to the published paper of Potter, 1995 ‘Overview of proposed mechanisms for the hypocholesterolomic effect of soy’ wherein soybean protein has been shown to have beneficial effects on preventing hyperlipidemic or hypercholesterolemic lesions, which cause arteriosclerosis and hypertension. Glycinin, which forms 25–35% of the total soy protein, is used in this invention to provide antihypertensive peptides.
Reference is made to the published paper of Yu, et al, 1996 “Effect of soybean hydrolysate on hypertension in spontaneously hypertensive rats” (J. Korean Soc. Food. Sci. Nutr. 25, 1031–1036) wherein they have demonstrated that a soybean hydrolysate exerted an inhibitory activity of ACE in vascular tissue in vivo and lowered systolic blood pressure in spontaneously hypertensive rats. The present invention uses an isolated protein fraction, glycinin, of soy protein and not total soy protein.
Reference may be made to the published paper of Shin et al, 1995 ‘Fractionation of angiotensin converting enzyme inhibitory peptide from soybean paste’ (Korean J. Food Sci. Technol. 27, 230–234) wherein it is demonstrated that a fraction of fermented soybean paste contains ACE inhibitory components and the fraction F53 exerts a strong inhibitory activity in vitro. The present invention utilizes a purified glycinin fraction of soy protein isolate.
Reference may also be made to Shin et al, 2001, in the published paper “His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo” (J. Agric. Food Chem., 49, 3004–3009) wherein the authors report they have isolated and identified the ACE inhibitory peptide of the F53 fraction and have confirmed the ACE inhibitory activity and blood pressure lowering activity of the purified peptide in vivo. They also show that the synthetic peptide His-His-Leu resulted in a significant pressure decrease in the aorta and triple injections of the peptide decreased the systolic blood pressure by 61 mm Hg. Reference is be made to Shin et al., U.S. Pat. No. 6,232,438, wherein a process is described for preparing a highly safe and active inhibitor having the formula His-His-Leu or His-Leu-Leu and physiologically acceptable salts thereof. The present invention however uses isolated glycinin and not soybean paste.
Reference may be made to Mimura et al, 1993, U.S. Pat. No. 5,243,027 wherein the ACE inhibitory peptides are produced by an acid hydrolysis of cell free extracts of a bacteria. The present invention uses enzymes to produce the hydrolysate and the source is a plant protein.
Reference may be made to the published papers of Maruyama and Suzuki, 1982 and Maruyama et al., 1985 (Maruyama, S and Suzuki, H, “A peptide inhibitor of angiotensin-I converting enzyme in the tryptic hydrolysate of casein.” Agric. Biol. Chem, 46, 1393, (Maruyama, S., Nakagomi, K., Tomizuka, N. and Suzuki, H. “Angiotensin-I converting enzyme inhibitor derived from an enzymatic hydrolysate of Casein. II Isolation and bradykinin potentiating activity on the uterus and ileum of rats” (Agric. Biol. Chem., 49, 1405) wherein they have demonstrated that a penta-peptide and hepta-peptide purified from a tryptic hydrolysate of casein inhibited ACE.
Wu and Ding, 2001 in their published paper ‘Hypotensive and physiological effect of angiotensin converting inhibitory peptides derived from soy protein on spontaneously hypertensive rats’ (J. Agric. Food Chem., 49, 501–505) have demonstrated in vivo hypotensive activity of soy-protein derived ACE inhibitory peptide powder at a dosage of 100 mg/kg of body weight/day/for 4-week feed in spontaneously hypertensive rats. Reference may also be made to the published paper of Wu and Ding, 2002 ‘Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides’ (Food Research International, 35, 367–375) that a soy protein alkaline hydrolysate after ultra-filtration had a IC50 value of 0.065 mg of protein/mL. Two of the most potent peptides were Ap-Leu-Pro and Asp-Gly with IC50 values of 4.8 and 12.3 μM respectively. The present invention however uses glycinin, a fraction of total soy protein and uses various proteases other than Alcalase.
Miyoshi et al, 1991 in their published paper ‘Structures and activity on angiotensin-converting enzyme inhibitors in an α-zein hydrolysate’ (Agric. Biol. Chem., 55, 1313–1318, Agric. Biol. Chem., 55, 1221, Agric. Biol. Chem., 55, 1407) isolated peptides from a thermolysin hydrolysate of α-zein, the major component of maize endospem protein and showed they inhibit ACE. The tripeptides Leu-Arg-Pro, Leu-Ser-Pro, Leu-Asn-Pro had IC50 values of 0.27, 1.7, 1.9 μM respectively. The hypotensive activity of the synthetic peptide Leu-Arg-Pro on spontaneously hypertensive rats indicated that the blood pressure was decreased by 15 mm Hg after a 30 mg/kg intravenous injection. The present invention uses glycinin, the storage protein of soyabean.
Reference may be made to yet another published paper by Sato et al., 2002 (J. Agric. Food Chem., 50, 6245–6252) wherein the isolation and identification of ACE inhibitory peptides from the Protease S ‘Amano’ digest of the brown seaweed, Wakame (Undaria pinnatifida) and investigation on the resistance of these peptides to gastrointestinal proteases in vitro is reported. The blood pressure of spontaneously hypertensive rats decreased after a single oral dose of 1 mg/kg of body weight. The present investigation uses proteases other than Protease S‘Amano’ to produce the hydrolysate. Furthermore the ACE inhibitory peptides are obtained from glycinin the major storage protein of soybean.
Reference may also be made to the paper published by Hsu et al., 2002 (J. Agric. Food Chem., 50, 6109–6113) wherein they report the peptic hydrolysates of dioscorin, the storage protein of the tuber yam (Dioscorea alata cv Tainong) exhibited ACE inhibition. The storage protein used in the present invention is from legumes and the enzymes used to prepare the hydrolysate are other than pepsin.
Further more several patents (U.S. Pat. Nos. 5,510,331, 5,106,834, 4,914,129, 4,889,869, 4,798,821, 4,758,584, 4,703,043, 4,385,050, 4,191,753, 4,013,791) describe the preparation of synthetic peptides and peptide analogs as ACE inhibitors. The present invention describes the process for an enzymatic hydrolysate of a plant protein as an ACE inhibitor.
Furthermore numerous patents on ACE peptides ranging from tri- to nonapeptides have been published: U.S. Pat. Nos. 5,449,661, 5,071,955, 4,692,459, 4,585,758, 4,512,979, 4,191,753, EP 174162; Japanese laid-Open Patent Publication Nos 5-2994844, 5-262790, 4-247100. However none relate to the use of glycinin as the source of antihypertensive peptides.
Enzymatic hydrolysis seems to be the most appropriate method for preparation of tailor made ACE inhibitory peptides, not only because of their large-scale commercial availability but also because of the high quality of such products. Such hydrolysates are complex mixtures of peptide species containing one or more active constituents in low concentration. Although most peptides that exhibit ACE inhibitory activity are those that are isolated and characterized, it is the extensive and expensive protocols that are limiting factors. However from the point of view of commercialization it will be the mixture of peptides, not a single purified peptide, which would be applied as a health-enhancing ingredient for use in ‘physiologically functional foods’.